The principle of solid phase oligonucleotide synthesis traces its history to work of Merrifield, Khorana and others in the 1950's and 1960's. The development of automated synthetic methods over the past decade has had a major impact in the fields of molecular biology and biological chemistry.
The stepwise synthesis of deoxyoligonucleotides generally involves the formation of successive diester bonds between 5'-hydroxyl groups of bound nucleotide derivatives and the 3'-hydroxyl groups of a succession of free nucleotide derivatives.
The synthetic process typically begins with the attachment of a nucleotide derivative at its 3'-terminus by means of a linker arm to a solid support, such as silica gel or beads of borosilicate glass packed in a column.
The ability to activate one group of the free nucleotide derivative requires that other potentially active groups elsewhere in the reaction mixture be "protected" by reversible chemical modification. The reactive nucleotide derivative is a free monomer in which the 3'-phosphate group has been substituted, e.g., by dialkylphosphoamidite, which upon activation reacts with the free 5'-hydroxyl group of the bound nucleotide or oligonucleotide to yield a phosphite triester. The phosphite triester is then oxidized to a stable phosphotriester before the next synthetic step.
The 3'-hydroxyl of the immobilized reactant is protected by virtue of its attachment to the support, and the 5'-hydroxyl of the free monomer can be protected by a dimethoxytrityl ("DMT") group in order to prevent self-polymerization. Also, a methyl group is usually used to protect the hydroxyl on the 3'-phosphate.
Additionally, the reactive groups on the individual bases are also protected. A variety of chemistries have been developed for the protection of the nucleoside exocyclic amino groups. The use of N-acyl protecting groups to prepare N-acylated deoxynucleosides has found wide acceptance for such purposes.
After each reaction excess reagents are washed off the column, any unreacted 5'-hydroxyl groups are blocked or "capped" using acetic anhydride, and the 5'-DMT group is removed using 80% acetic acid to allow the extended bound oligomer to react with another activated monomer in the next round of synthesis.
Finally, the fully assembled oligonucleotide is cleaved from the solid support and deprotected, to be purified by HPLC or some other method. The useful reagents and conditions for cleavage depends on the nature of the linkage. With ester linkages, as are commonly provided by linkage via succinyl groups, cleavage occurs at the same time as deprotection of the bases, by the use of concentrated aqueous ammonium hydroxide.
Synthetic methodologies that were in common use only a decade ago, such as the phospodiester method, are now largely obsolete. Today almost all synthetic oligonucleotides are prepared by solid phase phosphoramidite techniques. See generally T. Brown and D. Brown, "Modern machine-aided Methods of Oligonucleotide Synthesis", Chapter 1, pp. 1-24 in Oligonucleotides and Analogues, A Practical Approach, F. Eckstein, ed., IRL Press (1991).
The reagent most commonly used for the cleavage/deprotection of synthetic oligonucleotides is the concentrated aqueous ammonium hydroxide method. See, e.g., Protocol 5 of Brown and Brown, cited above. It can be seen that the time required for an ammonium hydroxide incubation is usually on the order of many hours, and generally involves overnight, heated incubation.
There has been a common perception that scientific efforts involving oligonucleotides, such as the human genome project which requires the synthesis of large number of oligonucleotide probes, will be hampered until even more elaborate, and expensive, tools are developed. Some scientists are beginning to realize, however, that significant strides can be made by carefully picking and choosing among existing technologies. This will be particularly true for those technologies that lend themselves to automation. See, e.g., "Leroy Hood: Thinking Big in Seattle", Science 264:206-209 (1994).
It would be particularly useful, for instance, to be able to accelerate the results presently achieved by the synthesis of oligonucleotides using today's reagents.